title: Cutting edge proteomics: benchmarking of six commercial trypsins.
abstract: Tryptic digestion is an important component of most proteomics experiments, and trypsin is available from many sources with a cost that varies by more than 1000-fold. This high-mass-accuracy LC-MS study benchmarks six commercially available trypsins with respect to autolytic species and sequence specificity. The analysis of autolysis products led to the identification of a number of contaminating proteins and the generation of a list of peptide species that will be present in tryptic digests. Intriguingly, many of the autolysis products were nontryptic peptides, specifically peptides generated by C-terminal cleavage at asparagine residues. Both porcine and bovine trypsins were demonstrated to be tyrosine O-sulfated. Using both a label-free and a tandem mass tag (TMT) labeling approach, a comparison of the digestion of a standard protein mixture using the six trypsins demonstrated that, apart from the least expensive bovine trypsin, the trypsins were equally specific. The semitryptic activity led to a better sequence coverage for abundant substrates at the expense of low-abundance species. The label-free analysis was shown to be more sensitive to unique features from the individual digests that were lost in the TMT-multiplexing study.
authors.affiliation: Department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre, DK-2650 Hvidovre, Denmark. jakob.bunkenborg@regionh.dk
authors.forename: Jakob
authors.initials: J
authors.lastname: Bunkenborg
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Espadas
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019985:Benchmarking; D002417:Cattle; D013058:Mass Spectrometry / Q000592:standards; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis*; D010449:Peptide Mapping; D011506:Proteins / Q000737:chemistry*; D059748:Proteolysis; D040901:Proteomics; D013552:Swine; D014357:Trypsin / Q000737:chemistry / Q000592:standards*; D014443:Tyrosine / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D011506:Proteins; D014443:Tyrosine; D014357:Trypsin